RESEARCH ARTICLE

A PIM-CHK1 signaling pathway regulates PLK1 phosphorylationand function during mitosisKevin Adam1,2,*, Maelle Cartel2,3,*, Mireille Lambert1,2,*, Laure David2,3, Lingli Yuan4, Arnaud Besson3,Patrick Mayeux1,2,‡, Stephane Manenti2,3,‡ and Christine Didier2,3,‡,§

ABSTRACTAlthough the kinase CHK1 is a key player in the DNA damageresponse (DDR), several studies have recently provided evidenceof DDR-independent roles of CHK1, in particular followingphosphorylation of its S280 residue. Here, we demonstrate thatCHK1 S280 phosphorylation is cell cycle-dependent and peaksduring mitosis. We found that this phosphorylation was catalyzedby the kinase PIM2, whose protein expression was also increasedduringmitosis. Importantly, we identified polo-like kinase 1 (PLK1) as adirect target of CHK1 during mitosis. Genetic or pharmacologicalinhibition of CHK1 reduced the activating phosphorylation of PLK1 onT210, and recombinant CHK1 was able to phosphorylate T210 ofPLK1 in vitro. Accordingly, S280-phosphorylated CHK1 and PLK1exhibited similar specific mitotic localizations, and PLK1 was co-immunoprecipitated with S280-phosphorylated CHK1 from mitotic cellextracts. Moreover, CHK1-mediated phosphorylation of PLK1 wasdependent on S280 phosphorylation by PIM2. Inhibition of PIMproteins reduced cell proliferation andmitotic entry, which was rescuedby expressing a T210D phosphomimetic mutant of PLK1. Altogether,these data identify a new PIM–CHK1–PLK1 phosphorylation cascadethat regulates different mitotic steps independently of the CHK1DDR function.

This article has an associated First Person interview with the firstauthor of the paper.

KEY WORDS: PIM2, CHK1, PLK1, Mitosis

INTRODUCTIONCheckpoint kinase 1 (CHK1, encoded by CHEK1) is a conservedserine/threonine protein kinase, widely known as a master regulatorof the DNA damage response (DDR) pathway. Phosphorylation ofits substrates, including p53, CDC25 or Wee1, leads to cell cyclearrest and the initiation of DNA repair, thus protecting against celldeath induced by genotoxic stress. CHK1 knockout mice areembryonic lethal (Lam et al., 2004; Liu et al., 2000), and Chk1+/–

mice exhibit hematopoietic defects (Boles et al., 2010). CHK1-deficient blastocysts and embryonic stem cells also show severe

proliferation defects as well as an impaired cell cycle checkpointresponse (Liu et al., 2000; Takai et al., 2000). Regulation of CHK1activity by the upstream kinase ATR occurs through thephosphorylation of CHK1 on S345 and S317. Phosphorylation atthese sites is considered to be a hallmark of CHK1 activation incells. In addition, CHK1 phosphorylates itself on S296 to achievefull activation. Beside these well-described activating mechanisms,CHK1 phosphorylation on S280 by the mitogenic serine/threoninekinases AKT, p90RSK, and PIM1 and PIM2 (PIM1/2), was alsorecently reported in different models, with diverse consequenceson CHK1 activity and its subcellular localization. Our own studiesin leukemic cells have suggested that PIM1/2-dependentphosphorylation of this residue is crucial for resistance to DNA-damaging agents (Yuan et al., 2014a) and for cell proliferation inunperturbed conditions (Yuan et al., 2014b).

It is now clear that CHK1 also plays important roles in cellfunctions that are independent of the DDR pathway, although thesepathways have not yet been fully established. CHK1 monitors DNAreplication during unperturbed S phase, and there is evidence for itsinvolvement in the control of replication origin firing, elongationand fork stability (Petermann et al., 2010; Guo et al., 2015). Inaddition to DNA replication, recent reports have documented theinvolvement of CHK1 in mitosis, particularly in regulating theinitiation, progression and fidelity of unperturbed mitosis (Krämeret al., 2004; Enomoto et al., 2009; Tang et al., 2006; Zachos et al.,2007). In line with this, CHK1 haploinsufficiency in mice results inmultiple mitotic defects such as cytokinetic regression andincreased binucleation (Peddibhotla et al., 2009). CHK1 depletionin primary mammary epithelial cells isolated from Chk1+/– micealso causes apparent defects in kinetochore function, activatesthe spindle assembly checkpoint, and eventually leads to mitoticcatastrophe (Peddibhotla et al., 2009). Moreover, CHK1 appears to beessential for normal mitotic progression in HeLa cells and is involvedin the proper alignment of chromosomes to the equatorial plane duringmetaphase, possibly via the regulation of PLK1, in order to inactivatethe spindle assembly checkpoint (Tang et al., 2006). Other reportshave described a functional interaction between CHK1 and the AuroraB kinase, leading to increased Aurora B kinase activity (Petsalakiet al., 2011; Zachos et al., 2007). Finally, the mitotic functions ofCHK1 also involve the histone-dependent transcriptional regulation ofCDK1 and its activating partner cyclin B1 (Shimada et al., 2008).Interestingly, CHK1 is also a target of cyclin-dependent kinase 1(CDK1). CHK1 can be phosphorylated at S286 and S301 by CDK1during mitosis, promoting its translocation from the nucleus to thecytoplasm during prophase (Enomoto et al., 2009).

Together, these studies provide evidence for the existence ofDNA damage-independent functions of CHK1 during mitosis;however, there are still many questions regarding the precisemechanisms involved and the identity of the molecular targets ofCHK1 in this context. In this study, we established that CHK1Received 13 November 2017; Accepted 15 June 2018

1Institut Cochin, INSERM U1016, CNRS UMR 8104, Universite Paris Descartes,75014 Paris, France. 2Ligue Nationale Contre le Cancer, equipe labellisee. 3CancerResearch Center of Toulouse, INSERM U1037, CNRS ERL 5294, Universite deToulouse, 31100 Toulouse, France. 4Department of Hematology, The SecondXiangya Hospital, Central South University, No.139 Renmin Middle Road, Furong,Changsha, Hunan 410011, China.*,‡These authors contributed equally to this work

§Author for correspondence (christine.didier@inserm.fr)

L.Y., 0000-0002-9293-6388; C.D., 0000-0003-3979-0743

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phosphorylation at S280 is strongly increased during mitosis andthat PIM2 is the kinase controlling this phosphorylation. In turn,S280-phosphorylated CHK1 is able to directly phosphorylate PLK1on its conserved T210 residue, leading to PLK1 activation. Takentogether, these data identify a novel pathway governing CHK1-mediated regulation of PLK1 activity during mitosis.

RESULTSCHK1 is a target of PIM2 during mitosisWe have previously established that interfering with CHK1phosphorylation on S280 influences the proliferation rate ofleukemic cells (Yuan et al., 2014a). In order to better understandthe function of this phosphorylation in the regulation of cellproliferation, we first asked whether CHK1 phosphorylation onS280 was regulated during the cell cycle. For this, we followed theP-S280 level of CHK1 during cell cycle progression by western blotanalysis of synchronization experiments. We used an antibodyagainst phosphorylated S280 that we first characterized for itsspecificity and efficiency by western blot and immunofluorescence(Fig. S1A-C). In H1299 cells released from a thymidine block afterserum starvation, the highest level of phosphorylation of S280 wasobserved during G2/M progression (Fig. 1A). Similar results wereobtained with the human osteosarcoma U2OS and the hTERT-immortalized foreskin fibroblast hTERT BJ cell lines (Fig. S1D,E).Accordingly, CHK1 phosphorylation on S280was strongly increasedin HeLa, U2OS and hTERT BJ cell lines blocked in mitosis withnocodazole (Fig. 1B and Fig. S1F). These data indicate that CHK1 ismaximally phosphorylated on S280 during mitosis, suggesting thatS280 phosphorylation of CHK1 may play an important role incontrolling its function during this phase of the cell cycle.We then asked which kinase was involved in this phosphorylation

event. The PIM, AKT and p90RSK family kinases have been reportedto directly phosphorylate this residue in various cell types (Puc et al.,2005; Li et al., 2012; Yuan et al., 2014a). Thus, we tested the impact ofinhibiting these kinases on CHK1 S280 phosphorylation in U2OScells blocked in mitosis with nocodazole. As seen in Fig. 1C,inhibition of PIM1 and PIM2 (PIM1/2) for 4 h almost completelyabolished S280 phosphorylation under these conditions, whereasinhibition of AKT only had a limited effect and MEK inhibition didnot significantly modify S280-phosphorylated CHK1 (P-CHK1)levels. In order to rule out any indirect effects, we verified that theinhibitors did not significantly induce cell death (TrypanBlue staining,Fig. S1G) after 4 h of treatment. These results were confirmed byusing RNA interference-mediated down-regulation of PIM1/2 in thesecells, which also dramatically decreased CHK1 S280 phosphorylation(Fig. 1D). Altogether, these results strongly suggest that PIM1/2 areimportant inducers of S280 CHK1 phosphorylation during mitosis.Cell cycle-dependent expression of PIM2 has never been

reported. Therefore, we performed synchronization experiments inUT-7 cells, which are known to express high levels of PIM2 (Adamet al., 2015), in order to assess the expression profile of PIM2 duringthe cell cycle. As shown in Fig. 1E, PIM2 protein levelsprogressively increased during S phase and peaked during mitosisin these experiments, correlating with histone H3 phosphorylationlevels. In contrast, PIM1 remained almost constant throughout thecell cycle. We then analyzed the ability of PIM2 to phosphorylateCHK1 on S280 in vitro, as we already established previously thatPIM1 is involved in this phosphorylation in leukemic cells (Yuanet al., 2014a). Recombinant GST-CHK1 protein was incubated withpurified PIM2 kinase in the presence of ATP (Fig. 1F), and CHK1phosphorylation on S280 was then detected by western blotting.These experiments showed that PIM2 directly phosphorylates CHK1

on S280 as pharmacological inhibition (SGI-1776) of recombinantPIM2 kinase blocked this phosphorylation. We confirmed theseresults byoverexpressing PIM2 inU2OS andHeLa cells, which led toa dramatic increase in CHK1 S280 phosphorylation (Fig. 1G). CHK1S280 phosphorylation levels were also significantly increased inmitotic HeLa cells expressing an inducible PIM2 kinase (Fig. 1H).Altogether, these data indicate that PIM2 is a mitotic kinase thatphosphorylates CHK1 on S280 during mitosis.

Subcellular localization of PIM2 and S280-phosphorylatedCHK1 during mitosisOur data describe for the first time that both PIM2 expression andCHK1 S280 phosphorylation are high during mitosis. We thereforeinvestigated the subcellular localization of PIM2 and CHK1 duringthis phase. Since endogenous PIM2 is barely detectable byimmunofluorescence, we used cells inducibly overexpressing theHaloTag-PIM2 fusion protein to study the subcellular localization ofPIM2 in nocodazole-treated HeLa cells. For these experiments, co-staining was performed with an antibody against PLK1, a well-established marker of the different mitotic steps (Strebhardt, 2010).Indeed, PLK1 localization has been well characterized during mitosis,with centrosome, kinetochore and midbody localization consistentwith its multiple mitotic functions. We detected PIM2 associated withspindle poles in metaphase, after which it then relocalized to theequatorial plane where spindle microtubules overlap in the midzone ascells go through anaphase, and finally, it was observed in the midbodyduring cytokinesis (Fig. S2A). Interestingly, apparent co-localizationof PIM2 and PLK1 was visible in these experiments, suggesting apossible functional link between these two proteins.

We then determined the distribution of S280-phosphorylatedCHK1 during mitosis in H1299 cells (Fig. S2B), after validation ofthe anti-P-S280 antibody used in these experiments (Fig. S1B).During metaphase, phosphorylated CHK1 was found at theperiphery of chromosome arms, as has already been described forthe CHK1 protein (Peddibhotla et al., 2009). During anaphase,P-CHK1 was concentrated at the spindle midzone, and duringcytokinesis, P-CHK1 accumulated at the midbody where it partlyco-localized with citron kinase (Fig. S2C).

Based on these data, we then performed immunolabeling ofP-CHK1 and PLK1 in U2OS (Fig. 2A) and HeLa cells (Fig. S2D),in order to investigate the subcellular localization of these twoproteins during mitosis. The same localization patterns describedabove were observed, wherein P-CHK1 localized to the peri-chromosomal layer (PCL) during prometaphase, whereas PLK1marked kinetochores. At this stage and during metaphase, bothpartners closely localized with each other (Pearson’s coefficients ofr=0.612 and r=0.860, respectively). At the onset of sister chromatidseparation during anaphase, PLK1 was translocated to the spindlemidzone and phosphorylated CHK1 foci partially co-localized withit. Finally, during cytokinesis, a small fraction of phosphorylatedCHK1 co-localized with PLK1 at the midbody (Fig. 2A). Tovalidate these observations, proximity ligation assays wereperformed to detect the co-localization of PLK1 with S280-phosphorylated CHK1 in U2OS cells treated or not withnocodazole. A significant proximity between the two proteins wasvisualized in nocodazole-treated U2OS cells, consistent with dataobtained on normal growing (asynchronous) HeLa cells where wespecifically visualized the mitotic cells (Fig. 2C). Similar resultswere obtained on UT-7 cells following nocodazole treatment(Fig. S2E). Together, these results indicate that a fraction of PIM2,P-CHK1 and PLK1 co-localize at distinct locations at differentstages of the mitotic process (Fig. 2 and Fig. S2).

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Fig. 1. PIM2 phosphorylates CHK1 inmitosis. (A) H1299 cells were synchronized by serum starvation followed by a thymidine block, and were then released atdifferent times. P-CHK1, CHK1 and actin levels were analyzed with the antibodies indicated. In parallel, cell cycle distribution after synchronization was evaluatedby FACS analysis (right). (B) S280 phosphorylation status of CHK1 in mitotic extracts of HeLa and U2OS cell lines. (C) Nocodazole (200 ng/ml)-treated U2OScells were incubated for their final 4 h of culture with inhibitors targeting PIM (SGI-1776, 5 µM), AKT (AKT inhibitor VIII, 4 µM) or MEK (PD 0325901, 250 nM),before lysing cells and immunoblotting with the antibodies indicated. (D) U2OS cells were transfected with siRNA against PIM1 and PIM2 or CHK1, or with controlsiRNA (CTL) for 24 h. After a further 16 h incubation in the presence of nocodazole (200 ng/ml), cells were processed for immunoblot analysis. Data arerepresentative of three independent experiments. (E) Double thymidine block (DTB) synchronization of UT7 cells. After release from the second thymidine block,cells were collected and cell extracts were analyzed for PIM1, PIM2, P-histone H3 and actin expression. Cell cycle progression is also shown in the insert on theright. (F) An in vitro kinase assay using PIM2 and CHK1 recombinant proteins was performed as described in the Materials and Methods, then CHK1phosphorylation was evaluated by immunoblotting. (G) The effect of PIM2 expression on CHK1 phosphorylation was evaluated in U2OS and HeLa cell linesinducibly expressing PIM2when treated with doxycycline (Dox, 2 µg/ml). (H) Phosphorylation of CHK1 following the induced expression of PIM2 in amitotic HeLacell line. P-CHK1/CHK1 ratios are shown beneath the western blot.

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CHK1 phosphorylates PLK1 on T210 during mitosisTo test the possibility that PIM2, P-CHK1 and PLK1 functionallyinteract during mitosis, we then asked whether CHK1 and/or PIMkinases can regulate PLK1 phosphorylation on T210, a hallmark ofPLK1 activation. RNA interference experiments performed innocodazole-blocked U2OS cells demonstrated that down-regulatingeither CHK1 or PIM1/2 strongly decreased PLK1 T210phosphorylation (Fig. 3A). Since cells are blocked withnocodazole in these experiments, it is unlikely that these effectson PLK1 phosphorylation are due to indirect cell cyclemodifications. We further verified that RNA interference did notsignificantly change cell cycle profiles (Fig. S3A) or cyclin Bexpression (Fig. 3A) in order to rule out these indirect cell cycledistribution effects. Furthermore, we did not detect any cell death inthese experiments (Fig. S3B). This observation was confirmed bypharmacological inhibition of CHK1 in H1299 nocodazole-treatedcells (Fig. S3C). We then asked whether preliminaryphosphorylation of CHK1 on S280 is necessary for itsphosphorylation of PLK1. For this, PIM2 and CHK1 were co-transfected into U2OS cells, and the effect on PLK1phosphorylation was monitored by western blotting. First of all,we noticed that CHK1 overexpression has an effect onoverexpressed PIM2 levels probably due to some transfectionissues. As shown in Fig. 3B, co-expression of PIM2 and CHK1induced an important increase in PLK1 phosphorylation on T210.In contrast, when a S280A (SA) mutant of CHK1 was transfectedinstead of wild-type CHK1, PLK1 phosphorylation was notmodified. These data indicate that phosphorylation of CHK1 onS280 is necessary for its kinase activity on PLK1. In addition, weverified that overexpression of wild-type CHK1 or the S280Amutant did not affect cell viability (Fig. S3D). The involvement ofPIM2 in PLK1 activation was confirmed by inducibly expressingPIM2 in U2OS and HeLa cells (Fig. S3E), which showed that PIM2expression led to an increase in PLK1 T210 phosphorylation. Wethen asked whether phosphorylation of PLK1 affected its co-localization with CHK1 in mitotic cells. For this we used proximityligation assays to detect the co-localization of CHK1 with T210-phosphorylated PLK1 (Fig. 3C), in U2OS cells treated or not withnocodazole. A close association between CHK1 and PLK1phosphorylated on T210 (red dots) was significantly increased innocodazole-treated cells. The association of PLK1 and CHK1during mitosis was also analyzed by co-immunoprecipitationexperiments with a CHK1 P-S280-specific antibody in U2OScells that had been co-transfected with CHK1 and PIM2, andblocked in mitosis by nocodazole treatment. In these cells, PLK1co-immunoprecipitated with phosphorylated CHK1, confirming aclose association between these two proteins in mitosis (Fig. 3D).To determine whether CHK1 can directly phosphorylate PLK1,

we then performed in vitro kinase assays. As shown in Fig. 3E,recombinant CHK1 was able to directly phosphorylate PLK1 onT210, while PIM2 did not. Interestingly, the presence of PIM2in these experiments did not change the ability of CHK1 tophosphorylate PLK1 on T210, suggesting that S280phosphorylation of CHK1 does not modify the catalytic activityof CHK1, but rather alters its capacity to phosphorylate PLK1through the regulation of its localization and/or interaction withother proteins. To elucidate clearly the role of P-CHK1 in ourexperiments and after MS/MS analyses (Fig. S4), we observed thatthe commercial recombinant CHK1 protein is alreadyphosphorylated on residue S280, in consequence, we producedwild-type and S280 mutant forms of CHK1. As shown in Fig. S3F,recombinant proteins used for in vitro kinase assay with one of its

well-defined substrates, CDC25C, presented good kinase activity(Fig. S3F). Then, we performed in vitro kinase assay withrecombinant CHK1 WT and mutant proteins, PIM2 and PLK1, asshown in Fig. S3G. The results confirm the role of CHK1 in thephosphorylation of PLK1 and suggest that the S280A mutant doesnot significantly affect the catalytic activity of CHK1. Altogether,these data identify a phosphorylation cascade involving PIM2,CHK1 and PLK1 during mitosis.

PIM2 and CHK1 govern the phosphorylation and behavior ofPLK1 substrates during mitosisFinally, we asked whether interfering with PIM2 or CHK1expression or activity modifies PLK1 function during mitosis inHeLa cells. For this, we followed the behavior of twowell-describedmitotic substrates of PLK1, EMI1 andWEE1. EMI1 is an anaphase-promoting complex/cyclosome (APC/C) regulator, and itsphosphorylation by PLK1 induces its proteasomal degradation(Margottin-Goguet et al., 2003; Hansen et al., 2007; Moshe et al.,2004). This is also the case for WEE1 kinase, a negative regulator ofCDK activity, whose phosphorylation by PLK1 induces itsdegradation (Watanabe et al., 2004). We found that when CHK1was down-regulated by RNA interference in HeLa cells, levels ofEMI1 and WEE1 increased (Fig. 4A). To reinforce theseobservations, we performed five independent experimentsincluding two siRNA sequences for CHK1, which also resulted ina significant accumulation of EMI1 and WEE1 proteins (Fig. 4B).Similarly, RNA interference-mediated down-regulation of PIM1/2also induced the accumulation of EMI1 in HeLa cells (Fig. 4C), anddecreased the phosphorylation of nucleophosmin (NPM1), anotherpreviously described substrate of PLK1 (Zhang et al., 2004).Moreover, treatment of siRNA-transfected cells with the proteinsynthesis inhibitor cycloheximide showed that WEE1 and EMI1degradation was delayed when CHK1 or PIM kinases weredownregulated, indicating that CHK1 or PIM1/2 have a negativeimpact (decreased half-life) on the stability of these two proteins(Fig. 4D). Then, using an inducible PLK1 expression system thatallows doxycycline-dependent expression of a constitutively activeform of PLK1 (T210D) in HEK 293T cells (3×Flag T210D), wecould significantly reverse the accumulation of WEE1 and EMI1proteins under CHK1 or PIM1/2 downregulation (Fig. 4E). Finally, tosubstantiate our observations, we evaluated PLK1 activity moredirectly. For this, we immunoprecipitated PLK1 from HeLa cellstreated with CHK1 (500 nM SCH900776) or PIM (5 µM SGI-1776)inhibitors for 16 h, and monitored PLK1 activity by estimating itscapacity to phosphorylate twowell-characterized substrates CDC25C(S75) (Gheghiani et al., 2017) and NPM1 (S4) (Zhang et al., 2004), inan in vitro kinase assay (Fig. 4F). These experiments showed thatpharmacological inhibition of PIM1/2 or CHK1 significantly impairsPLK1 activity. Altogether, these results show that PIM1/2 and CHK1kinases are involved in PLK1 regulation.

The PIM2-CHK1-PLK1 pathway is involved in cellularproliferationFinally, we found that the functional interference of PIM1/2 activityeither by siRNA or with a potent selective small-molecule inhibitor(SGI-1776) led to a significant inhibition of proliferation in HeLa,U2OS and H1299 cells (Fig. 5A,B). To further specificallyinvestigate the role of the PIM2-CHK1-PLK1 axis in mitoticprogression (G2/M transition), we used HEK 293T (3×Flag T210D)and the inducible PLK1 expression system previously described.These cells were synchronized in G2 by treating them with the CDKinhibitor RO3306 (10 µM) for 16 h, after which they were released

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in fresh medium supplemented or not with the SGI-1776 PIMinhibitor. After 7 h of culture in these conditions, the mitotic indexwas evaluated by flow cytometry to detect levels of phosphorylatedhistone H3. Inhibition of PIM1/2 kinase significantly reduced the

percentage of mitotic cells (from 60.03±3.33% to 21.9±1.01%), butthe concomitant expression of a constitutively active PLK1 mutant(T210D) in part compensated for PIM1/2 inhibition and increasedthe percentage of cells entering mitosis (to 34.9±1.05%) (Fig. 5C).

Fig. 2. Subcellular localization of S280-phosphorylated CHK1. (A) Representative confocal images of immunofluorescence staining for α-tubulin (greenchannel), PLK1 (red channel) and P-CHK1 (S280) (cyan channel) in U2OS cells. Selected high-magnification regions of interest are shown for anaphase andcytokinesis. (B) Proximity ligation assays using P-CHK1 and PLK1 antibodies on U2OS cells treated or not with 200 ng/ml nocodazole. DNAwas counterstainedwith DAPI. Z-stack images were acquired (n≥100 nuclei) by confocal microscopy and quantification of the images was performed with ZEN and ImageJ software.The distribution of foci per cell is shown on the right. For co-staining of P-CHK1 and PLK1: 0.324±0.047 foci/cell (mean±s.e.m.) from 182 nuclei analyzed undernormal growing conditions, and 8.775±0.703 foci/cell from 276 nuclei analyzed following nocodazole treatment. P-values were determined using the non-parametric t-test (****P<0.0001). (C) Proximity ligation experiments were performed using the P-CHK1 and PLK1 antibodies on HeLa cells. DNA wascounterstained with DAPI. Each antibody used was tested alone for their PLA signal [PLK1 (I); P-CHK1 (II)], then the species-specific secondary antibodies (PLAprobes) alone (III) and all the mix without PLA probes (IV). Z-stack images were acquired by confocal microscopy and quantification of the images performed withZEN and ImageJ software. The distribution of foci per interphasic versus mitotic cells is shown on the right. For co-staining with P-CHK1 and PLK1 antibodies:1.935±0.143 foci/cell from 139 nuclei analyzed for interphasic cells, and 15.680±2.020 foci/cell from 19 nuclei analyzed from mitotic cells. Statistical analyseswere performed as in B (****P<0.0001).

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These results suggest that the PIM2-CHK1 pathwayparticipates in PLK1 activation by controlling its phosphorylation.Taken together, our results indicate that PIM2 and CHK1 areupstream regulators of PLK1 function during mitosis andproliferation.

DISCUSSIONIn the present work, we have identified the existence of a novelsignaling cascade composed of the PIM2 and CHK1 kinases thatleads to activation of the serine/threonine kinase PLK1 duringmitosis. In addition to its well-described functions in the DNA

Fig. 3. CHK1 regulates PLK1 during mitosis. (A) U2OS cells were transfected with control siRNA or siRNA targeting PIM1/2 or CHK1 for 24 h, then arrested inmitosis by treatment with 200 ng/ml nocodazole for 16 h. Each fraction was subjected to immunoblotting with the indicated antibodies; actin served asloading control and was used for quantification. (B) U2OS cells were co-transfected with PIM2 and either wild-type CHK1 or CHK1 S280A mutant (SA) for 24 h,before being cultured for a further 16 h in the presence of 200 ng/ml nocodazole. Whole cell extracts were analyzed by western blotting with the correspondingantibodies. (C) Proximity ligation assays were performed using the CHK1 and P-PLK1 antibodies on U2OS cells treated or not with 200 ng/ml nocodazole.DNAwas counterstained with DAPI. Z-stack images were acquired (n≥100 nuclei) by confocal microscopy and quantification of the images was performed withZEN and ImageJ software. The distribution of number of foci per cell is shown on the right. For co-staining of CHK1 and P-PLK1: 1.556±0.341 foci/cell(mean±s.e.m.) from 160 nuclei were analyzed under control conditions, and 9.948±0.600 foci/cell from 154 nuclei analyzed following nocodazole treatment.P-value was determined using the non-parametric t-test (****P<0.0001). (D) U2OS cells were co-transfected with CHK1 and PIM2 for 24 h followed by overnightnocodazole treatment (200 ng/ml), then immunoprecipitation was performed with the phospho-S280 antibody. The expression of CHK1, P-CHK1 and PLK1was evaluated in the initial fraction (Input), the supernatant of the immunoprecipitation (SN) and the immunoprecipitated fraction (IP) by immunoblotting. (E) In vitrokinase assays were performed with PIM2, PLK1 and CHK1 recombinant proteins, and the levels of PLK1 phosphorylation evaluated by immunoblotting.

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Fig. 4. See next page for legend.

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damage response, several studies have described an independentrole for CHK1 in unperturbed cell cycle progression, particularlyduring mitosis. For instance, CHK1 can phosphorylate and enhancethe activity of Aurora B, promoting the functions of this mitotickinase in chromosome segregation and cytokinesis (Petsalaki et al.,2011; Zachos et al., 2007). A role for CHK1 in the regulation of theBubR1-Mad2-Cdc20 complex and PLK1 have also been described(Zachos et al., 2007; Chilà et al., 2013; Tang et al., 2006), althoughthese different studies have not provided a comprehensive picture ofthe general impact of CHK1 on mitotic progression. Here, we showthat CHK1 directly phosphorylates PLK1 on its conservedactivating T210 residue, and thereby positively regulates thediverse functions of PLK1 during mitosis. Since STK10, SLKand Aurora A have also been previously characterized as directmediators of PLK1 phosphorylation at T210 (Walter et al., 2003;Mac�urek et al., 2008; Seki et al., 2008), it remains to be clarifiedhow these different kinases coordinate their activities tophosphorylate and activate PLK1.Although CHK1 activation has been described as essentially

dependent on ATR-mediated phosphorylation on S345 and S317, itremains unclear whether this mechanism is necessary for its DDR-independent functions. Indeed, several lines of evidence suggestthat CHK1 can phosphorylate its substrates independently of ATR.For instance, during the G2/M transition, CHK1 is localized to thechromatin where it phosphorylates histone H3 on T11 to trigger thetranscriptional expression of the mitotic effectors CDK1 and cyclinB1, allowing the cells to enter into mitosis (Shimada et al., 2008). Inaddition to its phosphorylation on S345 and S317 by ATR, CHK1

can also be phosphorylated on S280 by AKT, p90RSK or PIM1/2 –serine/threonine kinases that are involved in the activation of themajor oncogenic signaling pathways PI3K-AKT, MEK-ERK andSTAT5, respectively. The first publication in this field reported thatS280 phosphorylation by AKT in PTEN-mutated cells inhibitedCHK1 function by sequestering the protein to the cytoplasmiccompartment (Puc et al., 2005). More recently, p90RSK was alsofound to phosphorylate CHK1 on S280 in response to growth factorstimulation of quiescent cells, but surprisingly, in this case itpromoted its nuclear accumulation and activation rather thaninhibition (Li et al., 2012). Finally, we recently documented thatPIM kinases are involved in the phosphorylation of this residue inleukemic cells, in which these kinases are often overexpressed andplay an important oncogenic role. In a previous study, we found thatthis phosphorylation had no detectable effect on CHK1 subcellularlocalization but did improve the DNA damage-dependent functionsof CHK1 (Yuan et al., 2014a), as well as its capacity to stimulateleukemic cell proliferation (Yuan et al., 2014b). The reason for theapparent discrepancies between these different models is not yetclear, but one possibility is that the pathways responsible for CHK1S280 phosphorylation and the resulting effects on its function differbetween cell types and, perhaps, according to growth conditions.Here, we provide an additional and more general account of CHK1regulation by its phosphorylation on S280, by demonstrating thatthis is an important parameter of CHK1 function during mitosis, andthat PIM1/2 kinases are responsible for this mitosis-specificphosphorylation in different cellular models. Moreover, oneimportant question that remains unresolved is the importance ofCHK1 kinase catalytic activation (S296 phosphorylation) versusS280 phosphorylation in terms of regulating changes in itssubcellular localization. In a previous work from Li et al. (2012),the authors did not observe any significant modulation of S296phosphorylation following S280 phosphorylation by p90RSKin vitro. In light of this result, we speculate that this residue mayact as a platform to recruit specific partners that may be involved inCHK1 activation and localization.

Although it has been previously involved in the regulation of cellcycle proteins, such as the CDK inhibitor p27Kip1 (Morishita et al.,2008), this is the first time to our knowledge that PIM2 has beendescribed as a mitotic kinase.We found that its expression increasedduring mitosis, implying that its activity also increased since PIM2is constitutively active and does not need post-translational(phosphorylation) activating modifications (Adam et al., 2015). Incontrast to PIM2, PIM1 kinase has been previously involved in theregulation of mitosis. PIM1 interacts with and phosphorylates thenuclear mitotic apparatus protein NuMa (Bhattacharya et al., 2002)in a process that is important for maintaining a stable complexbetween NuMA, dynein-dynactin and HP1β, which linkschromosomal kinetochores to spindle microtubules. In addition,PIM1 overexpression results in genomic instability by over-ridingthe mitotic spindle checkpoint (Roh et al., 2003). Finally, the co-localization of PIM1 with PLK1 has been described at thecentrosome and midbody (Van der Meer et al., 2014), suggestinga functional interaction between these two proteins for mitoticprogression. It is difficult, however, to correctly distinguish thespecific cellular functions of PIM1 or PIM2 since these two kinasescan compensate for each other in many circumstances.Understanding how PIM1 and PIM2 precisely regulate thedifferent mitotic steps at the molecular level, and how theymutually complement or compensate for each other, still remainsa challenging task. Our work implicates PIM2 as a regulator ofCHK1 during mitosis, and it will be interesting to determine

Fig. 4. PIM2 and CHK1 govern the phosphorylation of PLK1 substratesduringmitosis. (A) To check the efficiency of the PIM2, CHK1 and PLK1 axis,HeLa cells were transfected with control siRNA and siRNA targeting CHK1 for48 h. The corresponding fractions were then analyzed by immunoblotting withantibodies against the WEE1 kinase and EMI1, a major modulator of APC/Cactivity, whose stability is modulated via phosphorylation by PLK1. Actin wasused as a loading control. (B) Quantification of CHK1, WEE1 and EMI1 proteinlevel in HeLa cells transfected with control siRNA or siRNA targeting CHK1(two siRNA sequences were used) for 48 h, then analyzed by western blotting.Relative CHK1, WEE1 and EMI1 protein levels were normalized to actin, andset to 1 for control transfected cells. Data are mean±s.e.m. of five independentexperiments. For statistical analyses, non-parametric t-test was used(*P≤0.05, **P≤0.01 and ***P≤0.001). (C) Similar experiments were performedin HeLa cells treated with the indicated siRNA targeting PIM1 and PIM2kinases for 48 h. Cell extracts were analyzed by immunoblotting and wereprobed with the corresponding antibodies. Quantitative PCRwas carried out toevaluate the relative mRNA expression of PIM1 and PIM2 in theseexperiments. (D) HeLa cells were transfected with an siRNA control or siRNAtargeting CHK1 (two siRNA sequences were used) or siRNA targeting PIM1and 2 for 48 h, then cells were treated with cycloheximide (50 µg/ml) for theindicated times, finally subjected to immunoblotting. The relative WEE1 andEMI1 protein levels were normalized to actin, and set to 1 for untreated cells.Graph represents the quantification of EMI1 and WEE1 protein level at T4 forthe indicated conditions. Data are mean±s.e.m. of three independentexperiments. Statistical analyses were performed as in B. (E) HEK 293T cells(3×Flag T210D) inducibly expressing the PLK1 mutant T210D weretransfected with control siRNA, or siRNA targeting CHK1 or PIM1/2 for 24 hand then treated or not with doxycycline (200 ng/ml). Cell extracts weresubjected to immunoblotting using indicated antibodies. The relative level ofEMI1 and WEE1 was normalized to actin, and set to 1 for control transfectedcells. Data are mean±s.e.m. of six independent experiments. Statisticalanalyses were performed as in B. (F) HeLa cells were treated with CHK1(SCH900776-500 nM) or PIM (SGI-1776-5 µM) inhibitors for 24 h, and celllysates were used for immunoprecipitation of PLK1 protein.Immunoprecipitates were subjected to in vitro kinases assay with NPM orCDC25C recombinant proteins as described in the Materials and Methods.Phosphorylation levels were monitored by immunoblotting using indicatedantibodies.

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whether this function can also be performed by PIM1 in specificcellular contexts.The functional interaction between PIM1/2, CHK1 and PLK1

during mitosis has important implications in the field of cancerbiology and therapy. Indeed, it has been shown recently that theinhibition of PLK1, by either shRNA or the pharmacologicalinhibitor BI2536 in prostate cancer cells overexpressing PIM1,resulted in a dramatic inhibition of tumor progression. Moreover,compared with control cells, PIM1-overexpressing cancer cells aremore prone to mitotic arrest followed by apoptosis due to PLK1inhibition (Van Der Meer et al., 2014). These results suggest thatover-activation of this newly identified signaling pathway due to theoverexpression of one of its components could sensitize cancer cellsand lead to its inhibition. This may be of particular interest in thecase of acute myeloid leukemia (AML), since (i) PIM2 kinase iswidely overexpressed in AML and is considered to be a potentialtherapeutic target for this pathology (Tamburini et al., 2009); (ii) wereported that PLK1 is often overexpressed in AML (Renner et al.,2009); and (iii) its pharmacological inhibition blocks proliferationand induces apoptosis in leukemic cell lines, and dramaticallyinhibits the clonogenic potential of primary cells from patients.Finally, we recently identified CHK1 expression as an independentprognostic marker in AML (David et al., 2016), and we described its

regulation by PIM2 phosphorylation in these tumors (Yuan et al.,2014a; Yuan et al., 2014b). Altogether, these data suggest thattargeting the PIM2-CHK1-PLK1 pathway in AML may constitutean interesting alternative or may complement chemotherapies forthe treatment of these pathologies.

MATERIALS AND METHODSCell lines and treatmentsHuman non-small cell lung carcinoma H1299 cells were maintained in RPMIcontaining 10% fetal calf serum (FCS). Human bone osteosarcoma epithelialcells (U2OS), the human adenocarcinoma HeLa cell line, human hTERT-immortalized foreskin fibroblasts (hTERT BJ), and U2OS or HeLa cellsexpressing the HaloTag-PIM2 fusion protein, were cultured in DMEMsupplemented with 10% FCS and 1% penicillin-streptomycin (PS). Forsynchronization, human leukemic cells (UT-7) were grown in DMEMcontaining 10% FCS and 10 ng ml−1 GM-CSF at 37°C with 5% CO2. Thedoxycycline-dependent expression system allowing the induction of aconstitutively active form of PLK1 (T210D) in HEK 293T cells (3×FlagT210D) was kindly provided by Dr Laurent Créancier (Pierre FabreLaboratories, Toulouse, France). To establish the HaloTag-PIM2 fusionprotein-inducible system, a lentiviral expression vector pLenti PGK BlastDEST (Life Technologies) was modified by the insertion of the HaloTagcDNA (Promega) into the AgeI site. Then, the PIM2 isoform 2 cDNAsequence was placed in phase with HaloTag thanks to the Gateway system.

Fig. 5. The PIM2-CHK1-PLK1 axis regulates cellular proliferation. (A) HeLa, U2OS and H1299 cells were treated with PIM inhibitor SGI-1776 (5 µM) for 48 hthen cell numbers were assessed by Trypan Blue staining. (B) Similar experiments were performed with RNA interference against PIM1/2 in HeLa cellsafter transfection for 48 h. (C) The effect of PIM inhibition on mitotic entry in G2-synchronized HEK 293T cells, and compensation with a constitutively active formof PLK1 (T210D). Using HEK 293T cells (3×Flag T210D) inducibly expressing the PLK1 mutant T210D, cells were synchronized in G2 by treatment withCDK inhibitor RO3306 (10 µM) for 16 h in the presence or not of doxycycline (200 ng/ml). Immediately after release fromG2 synchronization, cells were cultivatedfor 7 h with or without doxycycline (200 ng/ml) and/or PIM inhibitor (SGI-1776; 5 µM) and 200 ng/ml nocodazole in fresh medium. The mitotic index was thenevaluated by FACS analysis (see schematic). Immunoblotting was carried out to control PLK1 T210D mutant expression. P-values were determined with thenon-parametric t-test (**P=0.003).

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The final construct was used to produce lentiviral particles to transduce HeLaand U2OS cells, and stable clones were selected by using 2 µg/ml blasticidin.

Cytokines, pharmacological inhibitors and reagentsHuman granulocyte-macrophage colony-stimulating factor (GM-CSF)was from Miltenyi Biotec (Bergisch Gladbach, Germany). Doxycycline,nocodazole, the CDK1 inhibitor (RO3306) and thymidine were obtainedfrom Sigma-Aldrich (France). The CHK1 inhibitor (SCH900776) waspurchased from Active Biochem (Cliniscience, France). The PIM (SGI-1776) and MEK (PD0325901) inhibitors were from Selleckchem(Euromedex, France). The AKT inhibitor VIII was from Calbiochem(Merck Millipore, France). Other reagents were purchased from Sigma-Aldrich (France). The protein synthesis inhibitor cycloheximide wasobtained from Sigma-Aldrich (France).

TransfectionFor siRNA transfection into HeLa or U2OS cells, Genome control pool non-targeting siRNA (Dharmacon) or CHK1 siRNA (Sigma-Aldrich) wastransfected using 8 μl INTERFERin (Polyplus) in 6-well plates, as describedby the manufacturer. For transfection of expression vectors, 2×106 U2OScells were resuspended in 100 μl Nucleofector Kit V, according to themanufacturer’s instructions (program x-001-Amaxa, Cologne, Germany).Experiments were performed 48 h after transfection.

ImmunoblottingThe procedures used for gel electrophoresis and immunoblotting have beendescribed previously (David et al., 2016). Briefly, cells were lyzed directly inNuPAGE LBS sample buffer, then sonicated and heated at 95°C for 5 min.Samples were subjected to electrophoresis in NuPAGE 4-12% Bis-Tris pre-cast gels (Life Technologies). CHK1 was detected with mouse monoclonalantibodies (clone G4, cat. no. sc-8408, Santa Cruz Biotechnology), PLK1 bymousemonoclonal antibodies (clones PL6/PL2, cat. no. 33-1700, Invitrogen),PIM2 by rabbit polyclonal antibodies (clone H-73, cat. no. sc-28778, SantaCruz Biotechnology), P-PLK1 by rabbit monoclonal antibodies (cloneD5H7,cat. no. 9062, Cell Signaling Technology), P-Bad (S112) by rabbit polyclonalantibodies (cat. no. 5284, Cell Signaling Technology), P-histone H3 (S10) byrabbit polyclonal antibodies (cat. no. 9706, Cell Signaling Technology),Cyclin B1 by mouse monoclonal antibodies (clone GNS1, cat. no. sc-245,Santa Cruz Biotechnology), P-Chk1 (S280) by rabbit polyclonal antibodies(cat. no. AP3069a, ABGENT), P-p44-p42 (Erk1/2, T202/Y204) by mousemonoclonal antibodies (clone E10; cat. no. 9106, Cell Signaling Technology),p44-p42 (Erk1/2) by rabbit polyclonal antibodies (cat. no. 9102,Cell Signaling Technology), P-RPS6 by rabbit monoclonal antibody (clone2F9, cat. no. 4856, Cell Signaling Technology); RPS6 by rabbit monoclonalantibody (clone 5G10, cat. no. 2217, Cell Signaling Technology); P-AKT(S473) by rabbit monoclonal antibodies (clone D9E, cat. no. 4060, CellSignaling Technology), AKT by rabbit polyclonal antibodies (cat. no. 9272,Cell Signaling Technology), Emi1 by rabbit polyclonal antibodies (cat. no.38-500, Zymed),WEE1 bymousemonoclonal antibodies (clone B11, cat. no.sc-5285, Santa Cruz Biotechnology), P-NPM (S4) by rabbit monoclonalantibodies (cat. no. 3520, Cell Signaling Technology), P-CDC25C (S75) byrabbit polyclonal antibodies were kindly provided by Dr Olivier Gavet(Gustave Roussy Cancer Campus, Villejuif, France), P-CDC25C (S216) byrabbit polyclonal antibodies (cat. no. 9528, Cell Signaling Technology),CDC25C by rabbit polyclonal antibodies (cat. no. sc-327, Santa CruzBiotechnology), NPM by mouse monoclonal antibodies (cat. no. AB10530,Abcam), P-NPM (S4) by rabbit polyclonal antibodies (cat. no. 3520, CellSignaling Technology), and actin bymousemonoclonal antibodies (clone C4,cat. no. MAB1501, Santa Cruz Biotechnology). All antibodies are used at1:1000 dilution. Secondary antibodies conjugated to HRP were used afterincubation with primary antibodies. Immunoreactive bands were visualizedby enhanced chemiluminescence (PI32209; Thermo Fisher Scientific) with aSyngene camera. Quantification of chemiluminescent signals was done withthe GeneTools software (v.1.4.0.0) from Syngene.

ImmunofluorescenceCells were fixed with 4% formaldehyde in 1× PBS at room temperature for15 min, then permeabilized with 0.5% Triton X-100 in 1× PBS, as

previously described (David et al., 2016). Briefly, cells were stained withthe indicated antibodies and slides were mounted with ProLong Goldantifade reagent containing 4′,6-diamidino-2-phenylindole (P36931 fromInvitrogen-Life Technologies). Images were acquired using a Zeiss confocalmicroscope (LSM780) and were subsequently processed using the ImageJor ZEN software packages.

For proximity ligation assays, cells were washed twice with 1× PBS, fixedwith 4% paraformaldehyde then permeabilized. Unspecific proteins wereblocked with 3% FBS containing 0.1% Triton X-100 in 1× PBS for 30 min atroom temperature. Cells were then incubated with either primary antibodiesagainst CHK1 (DSC-310, cat. no. C-9358, Sigma, 1:400) and P-PLK1 (cat. no.9062, Cell Signaling Technology, 1:200) or P-CHK1 (cat. no. AP3069A,ABGENT, 1:200) and PLK1 (cat. no. 37-7100, Zymed, 1:400). Then, cellswere incubated with the appropriate DNA-linked secondary antibodies(Duolink kit, Sigma), and PCR in situ amplification was performed usingthe PLA technology, according to the manufacturer’s instructions. The PLAsignal was detected with a Zeiss confocal microscope. A series of Z-stackconfocal microscopy images was taken and quantification of the images wascarried out using ImageJ software. The HaloTag System was used according tothe manufacturer’s recommendations in combination with the previouslydescribed immunofluorescence protocol.

Cell synchronization, apoptosis and cell cycle analysisCells were synchronized by serum starvation coupled with either a thymidineblock (2.5 mM), a double thymidine block (2.5 mM), or nocodazole treatment(200 ng/ml), then cell cycle distribution was analyzed. Briefly, cells wereharvested and fixed in ice-cold 70% ethanol at −20°C. Cells were thenpermeabilized with 1× PBS containing 0.25% Triton X-100, resuspended in1× PBS containing 10 µg/ml propidium iodide and 1 µg/ml RNase, andincubated for 30 min at 37°C. Staining for mitotic cells was conducted usingphospho-histone H3 (S10) antibodies (cat. no. 06-570, Merck Millipore,1:133). Apoptotic cells were detected with Annexin-V-FITC detection kitfromBDPharMingen (SanDiego, CA,USA) according to themanufacturer’sinstructions. Trypan Blue stain (GIBCO, LifeTechnologies, CA, USA) wasalso used to evaluate cell death. Data were collected on a MACSQuant VYBAnalyzer (Miltenyi Biotec, BergischGladbach, Germany) and analyzed usingFlowJo v.10 software.

In vitro kinase assaysHuman PIM2 (SignalChem), PLK1 (Enzo Life Sciences) and GST-CHK1(Enzo Life Sciences) recombinant proteins were incubated in 30 μl kinasebuffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.1 mM EDTA,0.01% Brig35, 2 mM DTT) at 30°C for 60 min. The potent selectivesmall-molecule inhibitor (SGI-1776) was used as control of the specificityof the PIM2 kinase activity. Analyses were performed by immunoblotting.

To generate expression vector permitting production of a fusion protein(GST), WT CHK1 sequence was cloned into the pGEX-4T2vector. Wegenerated CHK1 S280 mutated to Ala by using site directed mutagenesiswith forward primer 5′-cgagtcactgcaggtggtgtgtcagagtctccca-3′ and reverseprimer 5′-tgacacaccacctgcagtgactcggggcctttttgc-3′. Recombinant GST-CHK1 and GST-CHK1 S280A proteins, as well as the maltose bindingprotein-CDC25C recombinant protein were produced in bacteria, aspreviously described by Brezak et al. (2005).

HeLa cells were treated with CHK1 (SCH900776-500 nM) or PIM (SGI-1776-5 µM) inhibitors for 24 h, then cells were lysed in buffer containing25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EGTA, 1% NP-40,10 mM N-ethylmaleimide, protease inhibitor cocktail (Roche AppliedScience, Basel, Switzerland) and phosphatase inhibitor cocktails 2 and 3(Sigma-Aldrich). The lysates were used for immunoprecipitation of PLK1protein (mouse anti-PLK1, Cocktail, Invitrogen). Immunoprecipitates werewashed three times with lysis buffer and subjected to in vitro kinases assaywith NPM (Abcam, UK) or CDC25C (plasmid provided by OdileMondésert, ITAV, Toulouse, France). Phosphorylation levels weremonitored by immunoblotting using phospho-specific antibodies.

Quantitative PCR (qPCR)Total RNA was extracted from U2OS cells transfected with control siRNA,siRNA targeting PIM1 and PIM2 or siRNA targeting CHK1, using the

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RNeasy QIAGEN kit according to the manufacturer’s instructions. RNApurity and concentration were monitored with a NanoDrop ND-1000spectrophotometer (NanoDrop Technologies Inc., Thermo Fisher Scientific).cDNAs were synthesized from 1 μg total RNA using SuperScript III Reversetranscription (Invitrogen). Real-time qPCRs were performed on a StepOneReal-Time PCR System (Thermo Fisher Scientific) with a TaqMan GeneExpression Master Mix (Applied Biosystems). The primer used wasHs01065498_m1 for PIM1, Hs00179139_m1 for PIM2 and Hs00967506_m1 (Applied Biosystems) for CHEK1. GUSB (Hs00939627_m1) and B2M(Hs00984230_m1) were used as housekeeping genes. Results were analyzedwith the stepOnePlus software v.2.2.2 using the conventional ΔΔCt method.

NanoLC-MS/MS analysis and database searchesAfter SDS-PAGE separation of the in vitro kinase reaction, the silver-stainedcorresponding band was in-gel digested with trypsin and analyzed by onlineLC-MS analysis.

CHK1 LC-MS analysisThe peptides digested from CHK1 were measured on an SCIEX 5600+TripleTOF mass spectrometer operated in DDA mode. A Dionex Ultimate3000 nanoLC HPLC system and a Hypersil GOLD 150×0.32 mm column(Thermo Fisher Scientific), packed with C18 3 μm 175 Å material wereused for peptide separation. For the HPLC method, the buffer A used was0.1% (v/v) formic acid, and the buffer B was 0.1% (v/v) formic acid, 90% (v/v)acetonitrile. The gradient was 4-45% buffer B in 24 min with a flow rate of5 µl/min. For MS, a survey scan at the MS1 level (350-1600 m/z) was firstcarried out with 250 ms per scan. Then, the Top20 most intense precursors,whose charge states are 2-4 were fragmented. Signals exceeding 75 countsper second were selected for fragmentation and MS2 spectra generation.MS2 spectra were collected in the mass range 100-1600 m/z for 80 ms perscan. The dynamic exclusion time was set to 10 s.

LC-MS data analysisTo identify CHK1 peptides, profile-mode.wiff files from data acquisitionwere centroided and converted to mzML format using the AB Sciex DataConverter v.1.3 and submitted to Mascot (v.2.5) database searches againstUniProt SwissProt human database. ESI-Quad-TOF was chosen as theinstrument, trypsin/P as the enzyme and 2 missed cleavages were allowed.Peptide tolerances at MS and MS/MS level were set to be 20 ppm and0.5 Da, respectively. Peptide variable modifications allowed during thesearch were oxidation of M and phosphorylation of STY. To calculate thefalse discovery rate (FDR), the search was performed using the ‘decoy’option in Mascot.

Statistical analysisAt least three independent experiments were carried out to generate eachdataset and statistical analyses were performed with the Student’s t-testusing the Prism software package (GraphPad Software). Results areexpressed as means±s.e.m. Differences were considered significant for thefollowing P-values: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

AcknowledgementsWe thank Laetitia Ligat from the CRCT microscopy facility for helpful discussions onmicroscopy analyses. We gratefully acknowledge Dr Christine Dozier for criticalreview of the manuscript. The authors sincerely thank Romain Jugele for hisscientific contribution at the beginning of this project.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: L.Y., P.M., S.M., C.O.D.; Methodology: K.A., M.C., M.L., L.D.,S.M., C.O.D.; Software: M.C., L.D., C.O.D.; Validation: K.A., M.C., M.L., L.D.,C.O.D.; Formal analysis: K.A., M.C., L.D., S.M., C.O.D.; Investigation: M.C., M.L.,L.D., P.M., S.M., C.O.D.; Resources: P.M., S.M.; Data curation: K.A., M.C., P.M.,C.O.D.; Writing - original draft: K.A., P.M., S.M., C.O.D.; Writing - review & editing:M.C., A.B., P.M., S.M., C.O.D.; Visualization: S.M., C.O.D.; Supervision: P.M., S.M.,C.O.D.; Funding acquisition: P.M., S.M.

FundingThis research was funded by the Ligue Contre le Cancer.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.213116.supplemental

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RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs213116. doi:10.1242/jcs.213116

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